Repair of DNA damage is critical for the maintenance of genome integrity and cell survival. Living organisms have developed different pathways of DNA repair to deal with various types of DNA damage. One of the pathways of repairing DNA damage is through homologous recombination. The RecA protein of E. coli has been shown to be important for homologous recombination, and a great deal is known about this process in E. coli.
Although eukaryotic cells have RecA homologue(s), the mechanism of homologous recombination in vivo are poorly understood in eukaryotes. There is no evidence yet that eukaryotic cells employ RecA-like recombination mechanisms in vivo.
In the yeast S. cerevisiae, three epistasis groups of DNA damage-repair genes have been identified (Friedberg, E. C., Siede, W. and Cooper, A. J. (1991) in The Molecular and Cellular Biology of the yeast Saccharomyces (Broach, J. R., Pringle, J. R., Jones, E. W., eds) pp 147-192, Cold Spring Harbor Laboratory press, Plainview, N.Y.; Game, J. (1983) in Yeast Genetics: Fundamental and Applied Aspects (Spencer, J. F. T., Spencer, D., and Smith., A. R. W., eds) pp 105-137, Springer-verlag, New York.). The Rad52 epistasis group, which is mainly responsible for double-strand break (DSB) repair contains several genes: Rad50-Rad57, MRE11 and XRS2. Among these genes, mutations in Rad51, Rad52 and Rad54 cause the most severe and pleiotropic defects (Game, J. (1983) in Yeast Genetics: Fundamental and Applied Aspects (Spencer, J. F. T., Spencer, D., and Smith., A. R. W., eds) pp 105-137, Springer-verlag, New York.; Ajimura, M., Leem, S. H. and Ogawa, H. (1993) Genetics 133, 51-66; Ivanov, E. L., Korolev, V. G. and Fabre, F. (1992) Genetics 132, 651-664; Petes, T. D., Malone, R. E. and Symington, 1. S. (1991) 407-521.). Yeast strains lacking a functional Rad52 gene are extremely X-ray sensitive and deficient in mitotic and meiotic recombination (Resnick, M. A. (1969) Genetics 62, 519-531). It was reported recently that the overexpression of human Rad52 (HsRad52) conferred enhanced resistance to gamma rays and induced homologous intrachromosomal recombination in cultured monkey cells (Park, M. S. (1995) J. Biol. Chem. 270, 15467-15470). Mutations in different regions of Rad52 often result in different phenotypes (Boundy-Mills, K. and Livingston, D. M. (1993) Genetics 133, 39-49). It is proposed that the product of Rad52 gene is not required for the initiation of recombination, but is essential for an intermediate stage following the formation of DSBs but before the appearance of stable recombinants (Shinohara, A., Ogawa, H. and Ogawa, T. (1992) Cell 69, 457-470).
In the yeast S. cerevisiae, the major pathway of double-strand break repair is through gap repair, leading to gene conversion that may be associated with a crossover of flanking markers (Szostak, J. W., Orr-weaver, T. L., Rothstein, R. J. and Stahl, F. W. (1983) Cell 33, 25-35). Both Rad51 and Rad52 are important for the repair of breaks by this mechanism. However, gene conversion is only one of several homologous and non-homologous recombination pathways that are found in yeast and mammalian cells to repair chromosomal DSBs (Haber, J. E. (1995) BioEssays 17, 609-620). Based on transformation experiments in mammalian cells and in Xenopus oocytes, single-strand annealing, a non-conservative mechanism, has been proposed as an alternate pathway to repair double-strand breaks. (Fishman-Lobell, J., Rudin, N. and Haber, J. E. (1992) Mol. Cell Biol. 12, 1292-1303; Lin, F. -L. M., Sperle, K. and Sternberg, N. (1990) Mol. Cell. Biol. 10, 103-112; Maryon, E. and Carrol, D. (1991) Mol. Cell. Biol. 11, 3278-3287; Jeongyu, S. J. and Carrol, D. (1992) Mol. Cell. Biol. 12, 112-119.). Rad52 appears to be important for all homologous recombination events including gene conversion and single-strand annealing.
Despite the importance of Rad52 for homologous recombination the repair of chromosomal breaks, there is very little information available on the biochemistry of Rad52 protein. Homologs of Rad52 gene have been found in several eukaryotic organisms including yeast, mouse, chicken and human (see Park, J. Biol. Chem. 270:15467-15470 (1995); Muris et al., Mutation Res., DNA Repair 315:295-305 (1994); Bezzubova et al., Nucleic Acid Res. 21(25):5945-5949 (1993); Bendixen et al., Genomics 23:300-303 (1994); Shen et al., Genomics 25:199-206 (1995)).
Sequence analysis has revealed that N-terminal amino acid sequence of Rad52 protein is highly conserved while the C-terminal region is less conserved (Bezzubova, O., Schmidt, H., Ostermann, K., Heyer, W. D. and Buerstedde, J. -M. (1993) Nucleic Acids Res. 21, 5945-5949; Muris, D. F. R., Vreeken, K., Carr, A. M., Broughton, B. C., Lehman, A. R., Lohman, P. H. M. and Pastnik, A. (1993) Nucleic Acids Res. 21, 4586-4591; Bendixen, C., Sunjevaric, I., Bauchwitz, R. and Rothstein, R. (1994) Genomics 23, 300-303; Shen, Z., Denison, K., Lobb, R., Gatewood, J. M. and Chen, D. J. (1995) Genomics 25, 199-206).
It has been shown that Rad52 protein interacts through its C-terminal domain with the N-terminal domain of Rad51 protein in a species specific manner (Milne, G. T. and Weaver, D. T. (1993) Genes & Dev 7, 1755-176514; Donovan, J. W., T., M. G. and Weaver, D. T. (1994) Genes & Dev 8, 2552-2562; Shen, Z., Cloud, K. G., Chen, D. J. and Park, M. S. (1996) J. Biol. Chem. 271, 148-152). Similarly, a specific interaction with RPA has also been shown to be important for homologous recombination (see Park et al., J. Biol. Chem. 271(31):18996-19000 (1996)).
Yeast Rad52 protein has been reported to bind to both single and double-stranded DNA and carries out annealing of homologous single-stranded DNA. (Ogawa, T., Shinohara, A., Nabetani, A., Ikeya, T., Yu, X., Egelman, E. H. and Ogawa, H. (1993) Cold Spring Harbor Symp Quant. Biol. 58, 567-576.). Besides the report that human Rad52 protein interacts with human Rad51 protein and RPA, there are no other biochemical reports on the function of human Rad52 protein to date.